1. Field of Invention
This invention relates to a method of assaying the antioxidant capacity of a sample, especially mammalian serum or plasma, using a luminescent reaction.
2. Description of Prior Art
Cellular damage by free-radicals particularly oxy-radicals is generally believed to be a significant factor in many diseases including heart disease, cancer, rheumatoid arthritis and the aging process itself (Wayner, D.D.M. et al, Biochim. et Biophys. Acta (1987), 924, 408-419). It is known that blood including both cells and plasma has mechanisms to defend the organism against free-radicals. It is postulated that in some disease states these mechanisms are disturbed either by excessive production of free-radicals or by a decrease in the defence mechanism.
The mechanism is recognised as complex involving several substances capable of "scavenging". Such substances include urate, ascorbate, vitamin E, and various proteins including some containing selenium. There is experimental evidence (Sevanian, A. et al, J. Free Rad in Biol. Med. (1985), 1, 117-124), that the substances involved act in a combined manner to give a total anti-oxidant property to the system. It can be shown for example, that urate protects ascorbate (Sevanian et al, as above) and ascorbate protects other peroxyl radical scavengers (Frei et al. PNAS 86 6377 (1989).
To ascertain the total antioxidant capacity of blood plasma or serum, it is inconvenient, if not impossible, to determine the concentration of all the participating scavengers. Furthermore the total antioxidant capacity appears to be greater than the sum of those of all the participating scavengers (Sevanian et al., as above).
A suitable method for assaying rapidly and simply the total antioxidant capacity of biological fluids especially blood plasma or serum, would be of benefit as a diagnostic or patient monitoring tool.
A method for determining the total peroxyl radical trapping antioxidant activity of human blood plasma has been published (Wayner et al, Biochem. et Biophys. Acta (1987), 924, 408-419). The method is based on the discovery that peroxidation of aqueous dispersions of oxidizable organic compounds can be reproducibly initiated by thermal decomposition of the water-soluble azo compound 2,2'-azo-bis(2-amidinopropane hydrochloride (ABAP). At 37.degree. C. ABAP decomposes thermally to yield useful quantities of peroxyl radicals at a known and constant rate. The total peroxyl radical trapping antioxidant parameter (TRAP) is determined from the length of time a plasma sample resists peroxidation as measured by the rate of uptake of oxygen when subjected to attack by peroxyl radicals. Changes can be monitored by a pressure transducer or a modified oxygen electrode.
Although it has yielded useful information on the role of various constituents in plasma as antioxidants, this electrochemical method is complex and lengthy, taking up to 90 minutes per assay, which can only be done one at a time. It requires equipment which is not always available, is not easily operable and has to be cleaned after each assay. Accordingly, there is still a need for a suitable alternative, that is convenient to use and is capable of providing a total antioxidant capacity as well as information regarding the contribution of individual antioxidant components.
Further prior art is mentioned after the "Summary of the Invention", without which its context would not be clear.